The present invention relates to a process for preparing hydroxyoctadecadienoic acids, more particularly 9-hydroxy-10,12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid, and a mixture thereof, as well as the isomers thereof such as 9(R/S)-hydroxy-10E,12Z-octadecadienoic acid, optionally in the form of the esters thereof, more particularly in the form of the ethyl ester thereof.
In the description which follows, hydroxyoctadecadienoic acid is referred to as HODE.
The invention relates to a process for preparing HODE by controlled oxidation of linoleic acid and/or of linolenic acid, followed by reduction.
The invention also relates to a process for preparing HODE from triglycerides that are rich in linoleic acid and/or linolenic acid or from linoleic acid and/or lenolenic acid in the form of free fatty acid or in the form of an ester or salt, for example in the form of the ethyl ester.
The invention also relates to a drug, in particular for treating obesity, containing HODE as active principle and pharmacologically acceptable supports.
The invention also relates to a dietary supplement containing HODE, in particular for treating obesity.
The invention furthermore relates to a cosmetic product containing HODE and cosmetically acceptable supports, more particularly for the localized treatment of excess weight and its consequences on the appearance of the skin, such as xe2x80x9corange-peelxe2x80x9d skin, nodes of fat and steatomery.
It is generally accepted that the increase in the prevalence of excess weight and obesity in developed countries is due to an unsuitable diet, both as regards the excessive overall calorific intake and as regards an excessive fat content in this calorific intake. This very high fat content in the diet (estimated on average at close to 50% of the energy intake) may be reduced in the case of low-calorie diets in which the fats are greatly reduced.
However, the fats cannot be reduced to less than 30% of the energy intake since, not only do they play an important role in the texture and taste perception of foods, but also a minimum amount is necessary in the diet, in particular for the biosynthesis of essential fatty acids and hormones, and for the renewal of membrane phospholipids.
An essential axis of the research concerning the treatment of obesity has for several decades thus been the selection of xe2x80x9cgood fatsxe2x80x9d, i.e. fats which are not stored in the body, but used as a source of energy. In this context, it has been established that the nature of the fatty acids of which fats are composed plays a fundamental role in their metabolism. By way of example, short-chain fatty acids are more heat generating than long-chain fatty acids.
It has been possible to observe that the chain length of fatty acids has a direct influence on the energy expenditure and thus on their storage in the body.
One of the objects of the invention is to find a molecule for regulating the lipolytic activity of adipocytes, i.e. a molecule capable of hydrolyzing the triglycerides stored in adipocytes into free fatty acids and glycerols.
It is known that adrenaline and caffeine are two molecules that are known for their lipolytic effects. These are, in particular, molecules which increase the levels of cyclic adenosine monophosphate (cAMP), which is the first step in the activation of lipolysis.
One of the aims of the invention is thus, inter alia, to find a molecule which activates lipolysis to a greater extent than caffeine or adrenaline, by virtue of stimulating the production of cAMP by adipocytes.
Another aim of the invention is to provide a lipolytic molecule synthesized, simply and efficiently, from products commonly found in nature.
The inventors have discovered, surprisingly, that hydroxyoctadecadienoic (HODE) fatty acid derivatives have advantageous heat-generating properties without resulting in toxicity, and in particular 9-hydroxyoctadecadienoic acid in free acid form or in the form of esters or salts.
The invention thus relates to a process for obtaining an oil that is enriched in hydroxyoctadecadienoic (HODE) fatty acid and in particular in 9-hydroxyoctadecadienoic acid (9-HODE), or esters or salts thereof, from an oily mixture containing linoleic acid and/or linolenic acid, or esters or salts thereof, characterized in that a controlled oxidation of linoleic acid and/or of linolenic acid, or of esters thereof, is carried out in the presence of an oxidation catalyst, the oxidation being stopped when the total content of HODE, or of esters thereof, is greater than at least 5% and/or the content of 9-hydroxy-10,12-octadecadienoic acid (9-HODE) isomer, or of esters thereof, is greater than at least 1.5%, and in that the hydroperoxides formed during the oxidation reaction are reduced with a reducing agent.
The starting oily mixture is a plant oil containing more than 40% by weight of linoleic acid and/or of linolenic acid in the form of triglycerides, such as corn oil, borage oil, safflower oil, soybean oil, evening primrose oil, sunflower oil, blackcurrant pip oil, wheatgerm oil, hemp oil, marrow seed oil or flax oil.
The starting oily mixture is an oily mixture containing more than 40% by weight of linoleic acid and/or of linolenic acid, and preferably containing 75% ethyl linoleate.
The final content of total HODE or of 9-HODE in the enriched oil is at least 5%, or 1.5% respectively, by weight.
Preferably, the final content of total HODE or of 9-HODE in the enriched oil is respectively between 10% and 12% by weight and between 2.2% and 2.5% by weight.
The oxidation catalyst is an iron or copper halide, preferably FeCl3.
The reducing agent is preferably NaBH4.
The invention also relates to the oil enriched in hydroxyoctadecadienoic (HODE) fatty acid or esters or salts thereof, obtained as described above.
Furthermore, the invention relates to a drug for treating obesity, containing, as active principle, 9-hydroxyoctadecadienoic (9-HODE) fatty acid or pharmacologically acceptable esters or salts thereof, preferably in the form of enriched oil obtained as described above.
The invention also relates to a dietary supplement for treating obesity, containing, as active principle, 9-hydroxyoctadecadienoic (9-HODE) fatty acid or pharmacologically acceptable esters or salts thereof, preferably in the form of enriched oil obtained as described above.
The invention furthermore relates to a cosmetic product for treating obesity, containing, as active principle, 9-hydroxyoctadecadienoic (9-HODE) fatty acid or cosmetologically acceptable esters or salts thereof, preferably in the form of enriched oil obtained as described above.
According to the invention, hydroxyoctadecadienoic acid (HODE) is obtained by controlled oxidation of linoleic acid and/or of linolenic acid. These fatty acids are present in many plant oils.
For example, the following plant oils contain more than 40% linoleic acid in the form of triglycerides: borage oil, safflower oil, soybean oil, evening primrose oil, sunflower oil, blackcurrant pip oil, wheatgerm oil, hemp oil, marrow seed oil, flax oil.
According to a first embodiment of the invention, the HODE can be obtained from triglycerides that are rich in linoleic and/or linolenic acid. This process is advantageous since it allows the use of a natural plant oil which is thus entirely suitable for oral use, but has the drawback of considerably limiting the final content of 9-HODE isomer. Specifically, the fact that the linoleic and/or linolenic acid is in the form of glycerides limits the oxidation effect, which is the first step in the preparation of 9-HODE, but above all the linoleic acid content of about 40 to 50% among the mixture of fatty acids in the natural oil limits the final content of 9-HODE.
The structure of the starting material is a linoleic acid triglyceride or a linoleic acid ester of general formula (I) below: 
in which X is a triglyceryl residue or an Et (ethyl) residue or any other C1-C6 lower alkyl residue.
Such a compound has a double bond in position 9,10 and a double bond in position 12,13. Controlled oxidation produces, according to the invention, the compounds of general formulae (II) and (III) below: 
which is the glyceryl or alkyl ester of 9-hydroxy-10,12-octadecadienoic acid, or 
which is the glyceryl or alkyl ester of 13-hydroxy-9,11-octadecadienoic acid.
Controlled oxidation of (I) thus gives a mixture of isomers (II) and (III).
Linolenic acid exists in two forms: xcex1-linolenic acid (or (Z,Z,Z)-9,12,15-octadecatrienoic acid) and xcex3-linolenic acid (or (Z,Z,Z)-6,9,12-octadecatrienoic acid).
The oxidation reaction is carried out in the presence of an oxygenation catalyst such as an iron or copper halide, preferably FeCl3. The reaction has a duration ranging from 12 to 78 hours and a temperature of between xe2x88x9220xc2x0 C. and 40xc2x0 C., at atmospheric pressure and/or under reduced pressure. The degree of oxidation is monitored by different criteria:
assay of the hydroxides formed by gas chromatography;
change in the ultraviolet absorbance at 235 xc2x15 nm;
variation of the refractive index.
When the weight percentage of compound (II) (9-HODE) or the weight percentage of the mixture of compounds (II) and (III) reaches from 1 to 20%, the mixture is reduced, i.e. the hydroperoxides formed during the oxidation are reduced. An ethanolic solution of NaBH4 or an ethanolic solution of any other reducing agent is used, which is added portionwise to the reaction mixture. The temperature of the mixture is maintained between 4 and 25xc2x0 C. The reduction is stopped when the temperature becomes constant. The reaction medium is neutralized to pH 6-7 by adding an acid solution, preferably a solution of a pharmacologically and/or cosmetologically acceptable acid, for example citric acid.
Compounds (II) and (III) are extracted with hexane or any other organic solvent capable of dissolving them. The organic phase is washed with water until neutral.
The organic phase is then dried over anhydrous sodium sulphate or anhydrous magnesium sulphate.
The filtrate is then evaporated under vacuum.
The product obtained is a pale yellow oily liquid having a slightly aromatic odor. It has a total content of compounds (II) and (III) (determined by gas chromatography) of greater than 5% by weight and a content of compound (II) [9(R/S)-hydroxy-10E,12Z-octadecadienoic acid or 9(R/S)-hydroxy-10E,12Z-octadecadienoic acid ester] of greater than 1.5% by weight.
According to a second embodiment of the invention, which is preferred, the starting material is an oily mixture containing 75% by weight of ethyl linoleate.
The principle is the same as that described for the first embodiment of the invention. A selective oxidation of ethyl linoleate is carried out, followed by a reduction of the hydroperoxides formed and a purification by successive washes. A pale yellow oil of specific odor is obtained, containing about 2.5% by weight of 9-hydroxy-10E,12Z-octadecadienoic acid (in ethyl ester form) and 12% of a mixture of compounds (II) and (III) (in the form of ethyl esters). The hydroxyl function may be in position 9 or 13 and the double bonds in conjugated form at 10,12 (trans,trans and cis,trans) and in position 9,11 (trans,trans and cis,trans).